Acanthamoeba Spp Research Articles

Using Acanthamoeba spp. as a cell model to evaluate Leishmania infections.

Leishmaniasis represents a severe global health problem. In the last decades, there have been significant challenges in controlling this disease due to the unavailability of licensed vaccines, the high toxicity of the available drugs, and an unrestrained surge of drug-resistant parasites, and human immunodeficiency virus (HIV)-Leishmania co-infections. Leishmania spp. preferentially invade macrophage lineage cells of vertebrates for replication after subverting cellular functions of humans and other mammals. These early events in host-parasite interactions are likely to influence the future course of the disease. Thus, there is a continuing need to discover a simple cellular model that reproduces the in vivo pathogenesis. Acanthamoeba spp. are non-mammalian phagocytic amoeba with remarkable similarity to the cellular and functional aspects of macrophages. We aimed to assess whether the similarity reported between macrophages and Acanthamoeba spp. is sufficient to reproduce the infectivity of Leishmania spp. Herein, we analyzed co-cultures of Acanthamoeba castellanii or Acanthamoeba polyphaga with Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania braziliensis. Light and fluorescence microscopy revealed that the flagellated promastigotes attach to the A. castellanii and/or A. polyphaga in a bipolar and or random manner, which initiates their uptake via pseudopods. Once inside the cells, the promastigotes undergo significant changes, which result in the obligatory amastigote-like intracellular form. There was a productive infection with a continuous increase in intracellular parasites. However, we frequently observed intracellular amastigotes in vacuoles, phagolysosomes, and the cytosol of Acanthamoeba spp. Our findings corroborate that Leishmania spp. infects Acanthamoeba spp. and replicates in them but does not cause their rapid degeneration or lysis. Overall, the evidence presented here confirms that Acanthamoeba spp. have all prerequisites and can help elucidate how Leishmania spp. infect mammalian cells. Future work exposing the mechanisms of these interactions should yield novel insights into how these pathogens exploit amoebae.

Quantification of Acanthamoeba spp. Motility.

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential contamination of contact lenses after water exposure, the natural behavior of this pathogen remains elusive. Understanding Acanthamoeba's movement patterns can inform us about how it colonizes contact lenses and contaminates the patient's cornea. It is fortunate that Acanthamoeba spp. are visible via brightfield microscopy starting at 4x magnification. Previous techniques have been developed to quantify Acanthamoeba motility in regard to cytopathic effects or under-exposure to the electric field. Here, we describe a method to track and quantify Acanthamoeba spp. motility long-term (hours to days), which is a protocol applicable to multiple amoeba strains, surfaces, and nutritional status of amoeba. This procedure is germane to determining many core motility quantifications, such as distance, speed, confinement, and directionality, which are necessary to monitor different stages of infection, proliferation, or behavioral change.

Isolates of Acanthamoeba species in the marine environment in the Philippines.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs.

Brainstorm: A case of granulomatous encephalitis

Background: Free-living amoebas (FLAs) can cause severe and fatal central nervous system infections that are difficult to diagnose. Methods: We present the case of a 74-year-old immunocompetent woman admitted for focal neurological symptoms with enhancing lesions in the right cerebellar hemisphere. A first cerebral biopsy showed granulomatous inflammation, but no microorganisms were identified. After transient clinical improvement, she eventually deteriorated 4 months after initial presentation, with an MRI confirming multiple new masses affecting all cerebral lobes. Results: A second brain biopsy revealed granulomatous and acute inflammation with organisms containing a large central nucleus with prominent karyosome, consistent with FLAs. Immunohistochemical and polymerase chain reaction assays performed at CDC were positive for Acanthamoeba spp, confirming the diagnosis of granulomatous amoebic encephalitis (GAE) caused by Acanthamoeba spp. The patient was treated with combination therapy recommended by CDC, but died a few days later. Upon histopathological rereview, amoebic cysts and trophozoites were identified by histochemical and immunohistochemical methods in the first cerebral biopsy. Conclusion: FLA infections can be challenging to diagnose because of the low incidence, non-specific clinical and radiological presentation, lack of accessible diagnostic tools, and clinicians’ unfamiliarity. This case highlights the importance of recognizing FLA as a potential cause of granulomatous encephalitis, even in the absence of risk factors, as early treatment might be associated with favourable outcomes in case reports. When suspected, CDC laboratories offer tests to confirm the diagnosis promptly.

Free-living amoebic encephalitis - Case series.

Free-living amoeba is ubiquitous in fresh water, mud, and moist soil; although seldom pathogenic to humans, Naegleria fowleri, Acanthamoeba spp., and Balamuthia spp. are known to cause infections of the central nervous system. We report two interesting cases, both of which presented with a rapid-onset and fulminant course. The first case details a 36-year-old male with a history of surgically corrected nasal bone fracture 15 years back, who presented with acute onset of fever, headache and convulsions. Direct smears of cerebrospinal fluid (CSF) revealed motile trophozoites resembling N. fowleri and were later confirmed by molecular diagnosis (polymerase chain reaction [PCR]). Subsequently, the source was identified as water used for religious purposes. Unfortunately, the patient succumbed to cardiac arrest. The second case is that of a 4-year-old boy from Palakkad district, Kerala, who presented with sudden onset of nasal discharge, fever, headache, vomiting, seizures and altered sensorium. His CSF smear examination showed motile trophozoites resembling Acanthamoeba spp. While undergoing treatment, he seized thrice before going into cardiac arrest. Despite extensive resuscitation measures, the child could not be revived and passed away. The postmortem CSF sample sent for molecular analysis confirmed infection by Acanthamoeba spp. Rapid progression and lack of definite treatment options make this a highly fatal condition. Although clinical presentations of both patients were indicative of pyogenic meningitis, parasitic infection was suspected when the CSF was found turbid with no bacteria, high protein, and low sugar. High index of suspicion helped us to get an early preliminary diagnosis from direct microscopy.

Investigation of Healthcare-Associated Infection Risks from Ice: Summary of CDC Consultations 2016-2023

Background: Nonsterile ice is frequently used in healthcare settings for a wide array of patient care activities and clinical procedures. However, this ice can harbor pathogenic organisms which can threaten patient safety and cause outbreaks. We sought to characterize recent Centers for Disease Control and Prevention (CDC) consultations involving ice leading to healthcare-associated infections (HAIs). Methods: We reviewed internal CDC records from the Division of Healthcare Quality Promotion (DHQP) to identify investigations of outbreaks and potential outbreaks involving the use of ice in healthcare facilities. We searched records from January 1, 2016, through November 30, 2023, for keywords related to ice. We excluded consultations in which ice was not thought to be a potential transmission pathway as well as those in which only sterile ice products (e.g., saline slush) were investigated. Results: We identified 45 consultations for ice-related investigations, involving a total of 533 patients. Nontuberculous mycobacteria were the most frequently implicated organisms, appearing in 40% (n=18) of investigations. Eighty-four percent (n=38) of investigations occurred in acute care hospitals. The most frequently implicated hospital settings were intensive care units (13%, n=6), operating rooms (13%, n=6), and bronchoscopy suites (13%, n=6). We identified a variety of plausible exposure pathways, including direct ingestion of ice by patients, use of ice during the bronchoscopy procedure, use of nonsterile ice in heater-cooler devices during cardiothoracic surgery, and the use of ice to chill saline for respiratory care. Environmental sampling directly of ice machines was performed in 62% of investigations (n=28) and nonsterile ice from these machines was sampled in 9% of investigations (n=4). Among those investigations in which ice machines were sampled, the organism implicated in the outbreak was isolated in 54% of investigations (n=15). Among those investigations in which ice itself was sampled, the organism implicated in the outbreak was isolated in 75% of investigations (n=3). These organisms included Mycobacterium mucogenicum, Burkholderia multivorans, and Acanthamoeba spp. Conclusions: The use of nonsterile ice during clinical care is a potential source of pathogens that cause patient infections and HAI outbreaks. Healthcare personnel should be aware of the risk posed by nonsterile ice and consider avoiding its use, especially when caring for patients who are critically ill or immunocompromised. Healthcare facilities should ensure regular cleaning and disinfection of ice machines to decrease their microbial burden. When HAI outbreaks involving water-associated organisms are identified, nonsterile ice should be considered as a potential mode of transmission.

Can Acanthamoeba keratitis be properly diagnosed without culture in the real-world clinical microbiology laboratory?: a case report

Acanthamoeba species are ubiquitous, free-living organisms found in the environment. They can cause a sight-threatening cornea disease, termed Acanthamoeba keratitis, and are often misdiagnosed, causing delayed administration of the correct treatment. Herein, we report a case of Acanthamoeba keratitis diagnosed without culture. A 12-year-old girl with a history of wearing contact lenses presented with complaints of pain, irritation, and hyperemia in the left eye. Corneal scraping-smeared slide, and liquids with contact lenses were submitted to the clinical microbiology laboratory. Cultures of Acanthamoeba spp. were not available; thus, they were stained with calcofluor white. The isolation of Acanthamoeba from the corneal scraping allowed the detection of trophozoites and cysts based on their morphological characteristics. PCR targeting the 18s rRNA gene and subsequent sequencing revealed 99% identity with the Acanthamoeba spp. Although it is challenging to find real-world evidence of Acanthamoeba in clinical microbiology without using culture methods, this case underscores the need for clinical microbiology laboratories to maintain their inspection capabilities.

Amoebicidal Effect of COVID Box Molecules against Acanthamoeba: A Study of Cell Death.

Acanthamoeba spp. can cause a sight threatening disease. At present, the current treatments used to treat Acanthamoeba spp. Infections, such as biguanide-based antimicrobials, remain inefficacious, with the appearance of resistant forms and high cytotoxicity to host cells. In this study, an initial screening was conducted against Acanthamoeba castellanii Neff and murine macrophages J774A.1 using alamarBlue™. Among the 160 compounds included in the cited box, 90% exhibited an inhibition of the parasite above 80%, while only 18.75% of the compounds inhibited the parasite with a lethality towards murine macrophage lower than 20%. Based on the amoebicidal activity, the cytotoxicity assay, and availability, Terconazole was chosen for the elucidation of the action mode in two clinical strains, Acanthamoeba culbertsoni and Acanthamoeba castellanii L10. A fluorescence image-based system and proteomic techniques were used to investigate the effect of the present azole on the cytoskeleton network and various programmed cell death features, including chromatin condensation and mitochondria dysfunction. Taking all the results together, we can suggest that Terconazole can induce programmed cell death (PCD) via the inhibition of sterol biosynthesis inhibition.

Reliable and specific detection of Acanthamoeba spp. in dishcloths using quantitative real-time PCR assay

Acanthamoeba spp., are ubiquitous protist which belongs to Free-Living Amoeba (FLA) group, is considered as causal agent of side-threatening keratitis or fatal encephalitis among other human infections. Besides, this parasite has been reported as host for other microorganisms important to human health such as Campylobacter spp. or Vibrio spp. among others. This role of Acanthamoeba as pathogen and environmental phagocyte has increased the reports confirming its presence in human related environments, acting as a water quality indicator. Considering the tide relationship between water and kitchen environments, and the high prevalence of Acanthamoeba in water sources, the present study aims to establish a quick and accurate protocol based on DNA extraction and a real time qPCR assay to detect Acanthamoeba spp. in dishcloths. The procedure has been validated by processing 17 used dishcloths. Our findings demonstrated the high sensitivity of the qPCR assay used which was capable of detecting up to one Acanthamoeba from an in vitro contaminated dishcloth. The protocol accurately detected 64.7% of positive samples for Acanthamoeba spp, (in 4 samples DNA concentrations corresponded to 1-102 amoebae). Our findings demonstrate the importance of FLA surveillance by efficient and sensitive methods since one amoeba is capable of colonizing human related food environments such as kitchens sinks and could be a potential source of infection.

Determination of the anti-protozoal activity of medicinal agents using the phenomenon of plaque formation by Acanthamoeba spp.

Representatives of the genus Acanthamoeba are among the most widespread protists in the environment. They have a ubiquitous distribution and can sometimes cause quite serious pathologies in humans. The treatment ofp rotozoal infections caused by free-living amoebae is currently limited and often unsuccessful. In the presented investigation, amebicidal activity was determined against both the trophozoites and cysts of Acanthamoeba spp., which were isolated during the microbiological examination of environmental objects. The inhibitory activity of drugs in vitro was determined using the authors' proposed method, which is based on the plaque formation phenomenon: this is initiated by free-living amoebae when cultured in agar containing the bacteria Cellulosimicrobium sp. strain bent-1. Based on a series of experimental studies, the paper proposes a reliable and inexpensive method for determining the anti-protozoal activity of medicinal agents, which will significantly complement the current screening method system when studying existing drugs, or new drugs during their development stage.

Saline-Tolerant Pathogenic Acanthamoeba spp. Isolated from a Geothermal Power Plant.

Few studies have been conducted in the cooling systems of power plants; they have focused on Naegleria fowleri, leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic Acanthamoeba in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of Acanthamoeba was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 104 to 8.7 × 104 μS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. Acanthamoeba was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of Acanthamoeba culbertsoni killed the mice, having a mortality of 40 to 100%. Acanthamoeba genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.

First report of Acanthamoebae spp. isolation from a volcanic mud spring in the Philippines.

Acanthamoebae spp. is considered highly adaptive. The present study aims to establish the occurrence of free-living amoebae, particularly Acanthamoebae, to exist in extreme environments such as volcanic mud springs. Fifty surface water samples were collected from mud springs (34 samples), and flat rocks (16 samples) were collected, processed, and cultured. After 14 days of incubation, 32 (64%) plates showed positive amoebic growth. Nineteen (55.8%) of these plates came from the mud spring collection site, while 13 (81.2%) samples are from flat rock sources. DNAs from positive samples were made to react to polymerase chain reaction (PCR) using primer sets JDP1 5'GGCCCAGATCGTTTACCGTGAA-3' and JDP2 5'TCTCACAAGCTGCTAGGGAGTCA-3' for cells that resemble Acanthamoebae. Sequencing and basic local alignment search tool (BLAST) revealed a 99% similarity of isolates to Acanthamoebae spp. Identification of Acanthamoebae spp that can survive in higher temperatures is important public health information. The existence of such isolates in the environment has dire health implications, which suggests revisitation of water treatment protocols. Detection of such organisms in environmental sources used for recreational purposes provides information to local and international tourists who frequent them. This will result in the mitigation of potential future infection.

First report of free-living amoebae in watercourses in southern Brazil: molecular diagnosis and phylogenetic analysis of Vermamoeba vermiformis, Naegleria gruberi, and Acanthamoeba spp.

Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city.

In Vitro Stability of the Biological Activity of Voriconazole against Acanthamoeba castellanii

Acanthamoeba keratitis (AK) is a rare cornea disease caused by species of the Acanthamoeba genus. The antifungal voriconazole blocks the ergosterol synthesis in the protozoan membrane and is active against the cysts and trophozoites of Acanthamoeba spp. Due to the low stability of voriconazole, its options for eye drops are scarce. This study aimed to investigate the stability of the biological activity of voriconazole against two strains of Acanthamoeba castellanii and one clinical isolate from a patient with AK. To evaluate the stability of the biological activity of voriconazole, strains of A. castellanii (ATCC 50492) were exposed to different periods and voriconazole concentrations stored at 4 °C for 7, 15, and 30 days. The cytotoxicity assays were performed using SIRC (ATCC CCL-60™) cell line. The results indicated the amoebicidal effect of voriconazole against Acanthamoeba spp. within 24 h and 48 h of exposure, and the voriconazole solution was stable and retained antiamoebic activity when stored at 4 °C for up to 30 days. In the cytotoxicity test, the result demonstrated low cytotoxicity of the drug to the corneal rabbit cell line. However, there is a need to carry out further synergistic effects with other antiamoebic drugs and then in vivo experiments in the AK animal model.

Identification and genotyping of Acanthamoeba spp. in the water resources of western Iran

BackgroundAcanthamoeba spp. is opportunistic amoeba that resides in water, soil, and air. Some pathogenic genotypes of the genus of Acanthamoeba can cause granulomatous amoebic encephalitis (GAE) in people with a defective immune system. The parasite can also cause Acanthamoeba keratitis (AK) among contact lens users. This study was conducted to isolate and identify the Acanthamoeba genotypes in water resources in Lorestan province, western Iran. MethodsCollected 72 water samples from surface and groundwater (springs and aqueducts) in Lorestan province. Samples were filtered and cultured in non-nutrient 1.5% agar medium covered with Escherichia coli (E. coli) at 25 °C. DNA extraction was done and the PCR reaction was performed to detect the Acanthamoeba spp. The positive PCR products were sequenced to determine the genotypes of Acanthamoeba. ResultsOut of 72 examined water samples, 23.61% were positive for Acanthamoeba sp. by PCR. From PCR-positive samples, 8 (47.05%) samples were T4 genotypes and others were other Acanthamoeba genotypes (T1-T23). Therefore, approximately half of the genotypes belong to the pathogenic T4 genotype. ConclusionsThe water examined samples in western provinces of Iran have the potential risk factor for public health. Therefore, the efforts of healthcare providers are needed to identify, train, and prevention from human infections.

The Immunological Changes in the Skin of BALC/c Mice with Disseminated Acanthamoebiasis.

Toll-like receptors (TLR) are involved in the recognition of numerous pathogens, including Acanthamoeba spp. Thanks to this, it is possible for immune cells to recognize microorganisms and trigger the body's innate immune response. The stimulation of TLRs also leads to the activation of specific immunity. The aim of the study was to determine the TLR2 and TLR4 gene expression in the skin of BALC/c mice infected with Acanthamoeba with AM22 strain isolated from a patient. Receptor expression was assessed by real-time polymerase chain reaction (qPCR) in the amoeba-infected host with normal (A) and reduced immunity (AS) as well as in the control host with normal immunity (C) and reduced immunity (CS). Statistical analysis of TLR2 gene expression in A and AS groups compared to C and CS groups, respectively, were statistically insignificant. In the A group, we found statistical upregulation of TLR4 gene expression at 8 dpi compared to the C group. While in AS group, TLR4 gene expression was at a similar level, such as in the CS group. Taking into account the host's immune status, the TLR4 gene expression was statistically higher in the skin of host from A group than in host from AS group at the beginning of the infection. Increased TLR4 gene expression in hosts with normal immunity infected with Acanthamoeba suggests the involvement of the studied receptor in the course of acanthamoebiasis. The above research results provide new data on the involvement of the studied receptor in the skin in the host's immune defense triggered during the Acanthamoeba infection.

Molecular evidence of Monocercomonas and Acanthamoeba in the feces of captive reptiles

Reptiles are frequently kept as pet animals. They are considered as important reservoirs of protozoa with veterinary-medical significance. At a reptile farm in Ireland, fecal samples were collected from 98 captive reptiles, representing 43 species of three orders (Squamata, Testudines, and Crocodylia). After DNA extraction, all samples were screened by conventional PCRs, targeting the ribosomal small subunit (SSU) RNA and alpha-tubulin genes of trichomonads and SSU RNA gene of Acanthamoeba spp. One leopard gecko (Eublepharis macularius) was positive for a not yet reported species/genotype of the genus Monocercomonas, different from M. colubrorum. Various Acanthamoeba genotypes were detected in six reptilian species, i.e., Acanthamoeba genotype T11 in Eunectes notaeus and Heloderma suspectum/horridum; genotype T4 in Varanus exanthematicus, Chlamydosaurus kingii, and Macrochelys temminckii; and the genotype T13 in Iguana iguana. Some of these amoeba species might have clinicopathological significance in both humans and animals. Our findings highlight the importance to monitor pathogenic protozoa in pet as well as wildlife reptiles, as a source of possible infection for animals and humans living nearby.

Endoparasites Infecting Domestic Animals and Spectacled Bears (Tremarctos ornatus) in the Rural High Mountains of Colombia.

Simple SummaryThe spectacled bear (Tremarctos ornatus) is a threatened species, a member of the Ursidae family that lives in the Andes rural high mountain territories of Colombia, Venezuela, Ecuador, Peru, and Bolivia near livestock areas. Parasites in the spectacled bear are a relevant area of interest to preserve this species and understand its habitats and interactions with farm animals. The present work aimed to evaluate the presence of endoparasites in both T. ornatus and domestic animals in these areas, by copro- parasitological examination. The results indicate that some parasites have zoonotic potential in wild endangered species and domestic animals in Colombian regions. More sensitive molecular techniques are needed for further identification of the parasite species.This research described the co-infection prevalence of endoparasites in Tremartus ornatus and domestic animals in the rural high mountains of Colombia by copro-parasitological examination. Some parasites have a zoonotic potential in wild endangered species and domestic animals in Colombian regions. T. ornatus had a notable infection with Eimeria spp., Ascaris spp., Ancylostoma spp., and Baylisascaris spp. Cryptosporidium spp., Balantidium coli, Anoplocephala spp., and Acanthamoeba spp. In B. taurus, Eimeria spp. is coinfecting with Cryptosporidium spp. (6.6%) and represents 18% of the total parasitism. In E. caballus and B. taurus. Eimeria spp. coinfecting (34.7%), with the Strongylus spp. (21.9–25%). In T. ornatus, Eimeria spp. is coinfecting with Ancylostoma spp. (36.2%), Cryptosporidium spp., Ascaris spp., Baylisascaris spp., and B. coli.

Determination of human-pathogenic free living amoebas in drinking water supplies in Leicester, UK

Background and aims: Acanthamoeba spp. and Balamuthia mandrillaris are free-living amoebas (FLA) that can severely affect individuals. FLA are naturally resistant to harsh conditions such as pH, temperature and chemicals. Although we have previously reported the circulation of Acanthamoeba spp. and B. mandrillaris in open-water systems from Leicester (UK), little is known about their presence in public drinking water supplies. Thus, the aim was to identify potential risks due to their presence in tap water. Methods: Twenty-five tap water samples were collected in duplicate in March 2022 from public restroom facilities, located in parks (14), shopping/leisure centres (7), bus (2) and train (1) stations and a cemetery (1). Taps were appropriately cleaned before and between collecting the water samples. Samples were centrifuged at 2500rpm for 30 minutes for spore recovery. Pellets were suspended in phosphate-buffered saline to extract DNA by disrupting the spores using the Fast DNA® Spin kit and purified. A triplex real-time TaqMan PCR assay was performed for the simultaneous detection of FLA species, using established methodologies. Results: All fifty samples tested for FLA were negative. The drinking water supplies in the taps of Leicester’s public restrooms are not sourced from storage tanks, which reduce the overall risk of the presence of FLA in tap water. However, limitations in the collection may have affected our results, as the incidence of Acanthamoeba keratitis in the UK is much higher than in the United States and Netherlands. This higher incidence has been linked to the use of water storage tanks, which would be in line with our preliminary results. Conclusions: Regular monitoring of tap water quality would be required at a wider scale in the city including households, public institutions and hospitals in order to rule out the risks for the presence of human-pathogenic FLA, particularly Acanthamoeba spp.

Molecular identification and genotyping of Acanthamoeba spp., in bronchoalveolar lavage fluid from immunocompetent patients with chronic respiratory disorders (CRD).

This study aimed to investigate the presence and genotyping of Acanthamoeba spp., in the bronchoalveolar lavage fluid (BALF) of immunocompetent patients with chronic respiratory disorders (CRD). In this study, 211 BALF samples were collected from patients with CRD during the COVID-19 pandemic who were candidates for fiberoptic bronchoscopy (FOB) at Imam Khomeini Hospital, Sari, Mazandaran Province, northern Iran and investigated for Acanthamoeba spp., by PCR. A total of 211 FBAL samples were examined; 5 (5/211; 2.36%) were positive by using the PCR test for Acanthamoeba spp. According to sequence analysis, three strains belonged to the T4 genotype and one strain to the T2 genotype. Our data demonstrate that the presence of Acanthamoeba (T4 and T2) in BALF specimens of patients with respiratory infections. However, it is important to note that these findings may be merely accidental. Our findings suggest further investigation to fully understand the role of Acanthamoeba spp. in the pathogenesis of lung infections.